Lysozyme activity.

Lysozyme activity was measured as described previously (44). Briefly, a 2-mm-thick layer of agarose (0.9%) containing 50 mM sodium acetate (pH 4.5) and 0.5 mg/ml lyophilized cell wall from Micrococcus lysodeikticus (M3770; Sigma-Aldrich) was poured into sterile petri dishes, and several 4-mm-diameter holes were made. D. discoideum cells were grown in suspension, and 10⁸ cells were centrifuged (1,500 × g, 10 min) and then washed once with 5 ml of PB buffer (defined below). Each pellet was resuspended in 10 times its volume of 10% acetic acid containing protease inhibitors (5 mg/ml iodoacetamide, 47 μM leupeptin, 1.5 μM aprotinin, 100 μM phenylmethylsulfonyl fluoride) and was rotated on a wheel at 4°C overnight. Finally, the cell extract was centrifuged in an Airfuge (150,000 × g, 4°C, 15 min), the supernatant was collected and deposited (20 μl) into the holes on an agarose-Micrococcus plate, and the plate was incubated at 37°C for 24 h. Alternatively, purified ALFA-AlyL, ALFA-AlyA, ALFA-AlyAmut, and ALFA-AlyLmut tagged proteins were deposited (20 μl) into the holes. Lysozyme activity created a clear halo around the holes. The relative lysozyme activity in mutant cells was assessed by comparing the halo diameter with that created by applying various dilutions of WT extracts. Hen egg white lysozyme (Sigma) served as a control.